Deoxygalactonojirimycin derivatives

ABSTRACT

Novel N-alkyl derivatives of deoxygalactonojirimycin are provided in which said alkyl contains from 3-6 carbon atoms. These novel compounds are useful for selectively inhibiting glycolipid synthesis.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 08/102,654, filed Aug. 5,1993 and a continuation-in-part of application Ser. No. 08/061,645,filed May 13, 1993, now U.S. Pat. No. 5,399,567.

BACKGROUND OF THE INVENTION

This invention relates to novel N-alkyl derivatives ofdeoxygalactonojirimycin (DGJ) in which said alkyl groups contain from3-6 carbon atoms. These novel compounds are useful for selectivelyinhibiting glycolipid synthesis.

In applicants' application Ser. No. 08/061,645, filed May 13, 1993,certain N-aikyl derivatives of deoxynojirimycin (DNJ) are disclosed aseffective inhibitors of glycolipid biosynthesis. N-alkylated derivativesof DNJ were also previously known to be inhibitors of the N-linkedoligosaccharide processing enzymes, α-glucosidase I and II. Saunier etal., J. Biol. Chem. 257, 14155-14166 (1982); Elbein, Ann. Rev. Biochem.56, 497-534 (1987). As glucose analogues, they also have potential toinhibit glucosyltransferases. Newbrun et al., Arch. Oral Biol. 28,516-536 (1983); Wang et al., Tetrahedron Lett. 34, 403-406 (1993). Theirinhibitory activity against the glucosidases has led to the developmentof these compounds as antihyperglycemic agents and antiviral agents.See, e.g., PCT Int'l. Appln. WO 87/03903 and U.S. Pat. Nos.: 4,065,562;4,182,767; 4,533,668; 4,639,436; 4,849,430; 5,011,829; and 5,030,638.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel N-alkyl derivatives ofdeoxygalactonojirimycin (DGJ) are provided in which said alkyl containsfrom 3-6 carbon atoms and preferably from 4-6 carbon atoms. These novelcompounds are useful for selectively inhibiting glycolipid synthesis.The length of the N-alkyl chain has been found to be important to saidinhibitory activity since the non-alkylated DGJ and the N-methyl andN-ethyl derivatives of DGJ were each found to be inactive for suchinhibition. The N-propyl derivative of DGJ was partially active. Thus, aminimum alkyl chain length of 3 carbon atoms has been found to beessential for efficacy.

The biosynthesis of glycolipids in cells capable of producingglycolipids can be selectively inhibited by treating said cells with aglycolipid inhibitory effective amount of an N-alkyl derivative of DGJof the present invention. These inhibitory compounds can be used atconcentrations of about 10-fold less than the effective antiviralconcentrations of the corresponding N-alkyl derivatives of DNJ.Illustratively, the N-butyl DGJ is inhibitory of glycolipid biosynthesisat relatively low concentration of about 50 μM compared to the 0.5 mMlevel of concentration of N-butyl DNJ in cell culture systems forα-glucosidase I inhibition [Karlsson et al., J. Biol. Chem. 268, 570-576(1993).

The active N-alkyl derivatives of DGJ have a significant advantagesince, unlike the previously described N-alkyl derivatives of DNJ, theyselectively inhibit biosynthesis of glycolipids without effect either onthe maturation of N-linked oligosaccharides or lysosomalglucocerebrosidase. For example, in contrast to N-butyl DNJ, the N-butylDGJ of the present invention surprisingly does not inhibit theprocessing α-glucosidases I and II or lysosomal β-glucocerobrosidase.Likewise, the only prior reported experimental evidence usingdeoxygalactonojirimycin indicates that N-alkylation(N-heptyldeoxygalactonojirimycin) provides a modest increase in theaffinity towards certain β-glucosidases [Legler & Pohl, Carb. Res. 155,119 (1986)]. The inhibitory results described herein for the novelN-alkylated deoxygalactonojirimycin analoguss in which the alkylcontains from 3 to 6 carbon atoms were unexpected in view of thecorresponding activity of related iminosugar compounds.

Further uniqueness of the present invention is seen by the finding thatthe exemplary N-butyl and N-hexyl derivatives of DGJ completelyprevented glycolipid biosynthesis, whereas the N-butyl derivatives ofmannose, fucose and N-acetylglucosamine were without effect onglycolipid biosynthesis.

The inhibitory effect of these compounds on the biosynthesis ofglycolipids is illustrated herein in the myeloid cell line HL-60 and inthe lymphoid cell line H9. These are well-known, widely distributed andreadily available human cell lines. For example, HL-60 cells arepromyelocitic cells described by Collins et al., Nature 270, 347-349(1977). They are also readily available from the American Type CultureCollection, Rockville, Md., U.S.A., under accession number ATCC CCL 240.H9 cells are of lymphoid origin described by Gallo and Popovic, Science224, 497-500 (1984). They are also readily available from the samedepository under accession number ATCC HTB 176.

The inhibition of glycolipid biosynthesis by these N-alkyl derivativesof DGJ is further demonstrated herein by the reduction of the binding ofcholera toxin to the illustrative cell line H9 when cultured in thepresence on N-butyl DGJ. These compounds thus are also useful asanti-microbial agents by inhibiting the and bacterial toxins asillustrated hereinafter in Tables 1 and 2, respectively.

The inhibitory effect upon the biosynthesis of glycolipids is furtherillustrated by the ability of the N-butyl and N-hexyl derivatives of DGJto offset glucoceramide accumulation in a standard, state-of-the-art invitro model of Gaucher's disease. In this model, the murine macrophagecell line WEHI-3B was cultured in the presence of an irreversibleglucocerebrosidase inhibitor, conduritol β epoxide (CBE), to mimic theinherited disorder found in Gaucher's disease. WEHI-3B cells arewell-known, widely distributed and readily available murine macrophagecells. They are described in Cancer Res. 37, 546-550 (1977), and arereadily available from the American Type Culture Collection, Rockville,Md., under accession number ATCC TIB 68. The compounds of the inventionprevent lysosomal glycolipid storage which is useful for the managementof Gaucher's disease and other glycolipid storage disorders asillustrated hereinafter in Table 3. Gaucher's disease is an autosomalrecessive disorder characterized by an impaired ability to degradeglucocerebroside (glucosyl ceramide, Glc-Cer) due to mutations in thegene encoding β-glucocerebrosidase (β-D-glucosyl-N-acylsphingosineglucohydrolase, EC 3.2.1.45). This defect results in the lysosomalaccumulation of Glc-Cer in cells of the macrophage-monocyte system[Barranger and Ginns, in The Metabolic Basis of Inherited Diseases, ed.Scriver et al., pp. 1677-1698, McGraw-Hill, New York, (1989); Beutler,Science 256, 794-799 (1992)]. By slowing the rate of glycolipidsynthesis, the impaired catabolism of Glc-Cer can be offset, therebyleading to the maintenance of a balanced level of Glc-Cer.

The clinical management of Gaucher's disease currently relies uponeither symptomatic treatment of patients or enzyme replacement therapy[Beutler, Proc. Natl. Acad. Sci. USA 90, 5384-5390 (1993)]. In view ofthe prohibitive cost of enzyme replacement therapy and the requirementfor intravenous administration of glucocerebrosidase, an orallyavailable alternative therapy based around substrate deprivationconstitutes a useful alternative. The N-alkyl derivatives of DGJ arewater-soluble sugar analogs and, therefore, orally active. Since theN-alkyl DGJ compounds exhibit fewer complicating enzyme inhibitorycharacteristics than α- and β-glucosidase inhibitors, they alsoconstitute a preferable alternative to the N-alkyl DNJ compounds fortherapeutic management of Gaucher's disease and other glycolipid storagedisorders. The N-alkyl DGJ may also be used in combination withglucocerebrosidase for the treatment of Gaucher's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming theinvention, it is believed that the invention will be better understoodfrom the following illustrative detailed description of the inventiontaken in conjunction with the accompanying drawings in which:

FIG. 1 shows by one dimensional thin layer chromatography (1D-TLC) acomparison of N-alkylated imino sugars as inhibitors of glycolipidbiosynthesis. 1D-TLC separation was made of HL-60 total cellular lipidslabelled with [¹⁴ C]-palmitic acid. Cells were treated with either 0.5mM N-butyl deoxynojirimycin (NB-DNJ), N-butyl deoxymannojirimycin(NB-DMJ), N-butyl deoxygalactonojirimycin (NB-DGJ) or N-butyl2-acetamido-1,2,5-trideoxy-1,5-imino-D-glucitol (NB-NAG) or untreated(UT). Glycolipid biosynthesis inhibition was detected by the lack ofGlc-Cer, gangliosides and an unknown species (indicated with arrows).Glc-Cer migration was confirmed by inclusion of a [¹⁴ C]-Glc-Cerstandard on the TLC. The radiolabelled lipid species were visualized byautoradiography.

FIG. 2, in three parts, FIG. A, FIG. B and FIG. C, shows 2D-TLC analysisof HL-60 cells treated with either NB-DNJ or NB-DGJ. 2D-TLC separationwas made of total HL-60 lipids labelled with [¹⁴ C]-palmitic acid. Cellswere treated with either 0.5 mMNB-DNJ or NB-DGJ or untreated (UT).Lipids were assigned as follows (untreated cells, lefthand panel, FIG.2A): 1, gangliosides; 2, lysophospatidylcholine; 3, ceramidephosphorylcholine; 4, ceramide phosphorylethanolamine; 5,phospatidylcholine; 6, phosphatidylinositol; 7,phosphatidylethanolamine; 8, phosphatidylglycero1; 9,diglycosylceramide; 10, monoglycosylceramide; 11, cholesterol/fattyacids/neutral lipids; N and N*, are unknowns; and 0 is the sampleorigin. Following NB-DNJ and NB-DGJ treatment (middle and righthandpanels, FIGS. 2B and 2C, respectively) species 1 (gangliosides); 9(diglycosylceramide); 10 (monoglycosylceramide) and N*, (unknown) wereabsent. The radiolabelled lipids were visualized by autoradiography.

FIG. 3 shows the dose dependent effects of NB-DNJ and NB-DGJ onglycolipid biosynthesis. 1D-TLC analysis was made of total cellularlipids. HL-60 cells were labelled with [¹⁴ C]-palmitic acid in thepresence or absence (UT) of NB-DNJ or NB-DGJ at the indicatedconcentrations (μM). The migration position of [¹⁴ C]-Glc-Cer isindicated by arrows. The lipids were visualized by autoradiography.

FIG. 4, in two parts, FIG. A and FIG. B, shows the effects of increasingDNJ and DGJ N-alkyl chain length on inhibition of glycolipidbiosynthesis. 1D-TLC analysis was made of total cellular lipids. HL-60cells were treated with [¹⁴ C]-palmitic acid in the presence or absence(UT) of either DNJ, or the N-ethyl, N-methyl, N-propyl, N-butyl andN-hexyl derivatives of DNJ (lefthand panel, FIG. 4A) or DGJ, or theN-ethyl, N-methyl, N-propyl, N-butyl and N-hexyl derivatives of DGJ(righthand panel, FIG. 4B) at 0.5 mM concentration. The migrationposition of [¹⁴ C]-Glc-Cer is indicated with arrows. The lipids werevisualized by autoradiography.

FIGS. 5 and 6 show the analysis of NB-DNJ and NB-DGJ in an in vitroGaucher's disease model. Specifically, FIG. 5 shows the 1D-TLC analysisof glycolipids from WEHI-3B cells treated with either NB-DNJ or NB-DGJ,at the indicated concentrations (μM), and visualized by chemicaldetection (see methods hereinafter).

FIG. 6, in eight parts, FIGS. A,B,C,D,E,F,G,H, shows the transmissionelectron microscopy of WEHI-3B cell lysosomes: FIG. 6A, untreated; FIG.6B, conduritol β epoxide (CBE) treated; FIG. 6C, CBE and 500 μM NB-DNJ;FIG. 6E, CBE and 50 μM NB-DNJ; FIG. 6G, CBE and 5 μM NB-DNJ; FIG. 6D,CBE and 500 μM NB-DGJ; FIG. 6F, CBE and 50 μM NB-DGJ; FIG. 6H, CBE and 5μM NB-DGJ. The scale bar at the lower right hand corner of FIG. 6H isapplicable to all of FIGS. 6A through H and represents 0.1 μM.

FIG. 7 shows the effect of NB-DGJ on N-linked oligosaccharideprocessing. Specifically, it shows Endo H sensitivity of gp120 expressedin Chinese hamster ovary (CHO) cells in the presence or absence (-) ofeither NB-DNJ or NB-DGJ (0.5 mM and 5 mM). The arrows indicate themolecular weight of the untreated gp120 (120 kDa) and post endo Hdigestion (60 kDa). An additional band of low molecular weight(approximately 60 kDa) was present in some lanes and is a non-specificprotein precipitated by the solid phase matrix.

FIG. 8 is a graphical representation that shows, in three parts, FIG. A,FIG. B and FIG. C, the effect of imino sugar analogues on glycolipid andglycoprotein metabolizing enzyme activity. Enzyme activity wasdetermined in the presence of the following test compounds: DNJ, (♦);NB-DNJ, (▪); DGJ, (▴); NB-DGJ, () at concentrations shown (see methodshereinafter). FIG. 8A, UDP-glucose:N-acylsphingosineglucosyltransferase; FIG. 8B, β-glucocerebrosidase; FIG. 8C, processingα-glucosidase. Enzymatic activity is expressed as a percentage ofcontrol reactions that contained no test compound.

FIG. 9 shows the laser desorption mass spectrometry of N-butyldeoxygalactonojirimycin with a molecular weight of 220 (M+H) andobtained in greater than 95% purity.

FIG. 10 shows the ¹ H NMR spectrum of N-butyl deoxygalactonojirimycin.

FIG. 11 is a graphical representation of a cholera toxin binding assayand shows on the y-axis the % reduction in cholera toxin binding sitesper cell for H9 cells in which the cholera toxin was fluoresceinconjugated and in which the levels of binding to the cell surfaces ofuntreated (ut) cells and cells treated with N-butyldeoxygalactonojirimycin (NB-DGJ) or, for comparison, N-butyldeoxynojirimycin (NB-DNJ), at various levels shown on the x-axis(mg/ml), were measured by flow cytometry.

In order to further illustrate the invention, the following detailedexamples were carried out although it will be understood that theinvention is not limited to these specific examples or the detailstherein.

EXAMPLES Materials & Methods

Compounds:

N-Butyldeoxynojirimycin (NB-DNJ) was obtained from Searle/Monsanto (St.Louis, Mo., U.S.A.). Deoxygalactonojirimycin (DGJ), deoxyfuconojirimycin(DFJ), deoxymannojirimycin (DMJ), and2-acetamido-1,2,5-trideoxy-1,5-imino-D-glucitol (NAG), were obtainedfrom Cambridge Research Biochemicals (Northwich, Cheshire, U.K.). DGJ,DFJ, DMJ and NAG were reductively N-alkylated in the presence ofpalladium black under hydrogen using the appropriate aldehyde byconventional procedure as described by Fleet et al., FEBS Lett. 237,128-132 (1988). The reaction mixture was filtered through Celite and thesolvent removed by evaporation under vacuum. The resulting N-alkylatedanalogues were purified by ion-exchange chromatography (Dowex® AG50-X12,H+ form) in 2M NH₃ (aq) and the solvent removed by evaporation. Thesecompounds were lyophilised and analysed by 1D ¹ H NMR at 500 MHz on aVarian Unity 500 spectrophotometer and by matrix assisted laserdesorption (Finnegan). All compounds synthesised were greater than 95%pure. The following are representative examples of the synthesis of theforegoing N-alkylated compounds as used hereinafter.

Example 1

In a representative example of the preparation of the N-butyldeoxygalactonojirimycin, 30 mg (184 μmol) of deoxygalactonojirimycin wasdissolved in 1 ml of 50 mM sodium acetate buffer, pH 5.0, to which 20 mgof palladium black was added. A hydrogen atmosphere was maintained inthe reaction vessel and 100 μl (1.1 mmol) of butyraldehyde wasintroduced. The reaction was stirred for 16 hr. at room temperature (ca.20° C.). The reaction was stopped by filtration through a bed (1 g) ofCelite (30-80 mesh) and the reaction products were separated bychromatography using a column containing 4 ml of packed Dowex® AG50-X12(H+ form) resin. The N-butyl deoxygalactonojirimycin was eluted from thechromatography column with 2M ammonia. Its molecular mass was 220 (M+H)as determined by laser desorption mass spectrometry and its chemicalstructure was confirmed by 1D ¹ H NMR as shown in FIGS. 9 and 10,respectively.

Example 2

The synthesis procedure and compound analysis of Example 1 was repeatedexcept that caproaldehyde was substituted for an equivalent amount ofbutyraldehyde for analogous preparation of N-hexyldeoxygalactonojirimycin. Its molecular mass was 248 (M+H) as determinedby laser desorption mass spectrometry and its chemical structure wasconfirmed by 1D ¹ H NMR.

Example 3

The synthesis procedure and compound analysis of Example 1 was repeatedexcept that propanoyl aldehyde was substituted for an equivalent amountof butyraldehyde for analogous preparation of N-propyldeoxygalactonojirimycin. Its molecular mass was 206 (M+H) as determinedby laser desorption mass spectrometry and its chemical structure wasconfirmed by 1D ¹ H NMR.

The N-alkylated deoxygalactonojirimycin compounds prepared in theforegoing illustrative Examples 1 to 3 were obtained in overall yieldsof 68-74% based on the starting deoxygalactonojirimycin and were greaterthan 95% pure.

Enzymes and Enzyme Assays:

Porcine liver α-glucosidase I and rat liver α-glucosidase II werepurified to homogeneity and assayed by conventional procedure using a[¹⁴ C]-glucose labelled Glc₃ Man₉ GlcNAc₂ substrate as previouslydescribed by Karlsson et al., J. Biol,. Chem. 26.8, 570-576 (1993).

β-D-Glucosyl-N-acylsphingosine glucohydrolase (glucocerebrosidase) wasisolated from human placenta and purified to homogeneity according topublished standard methods [Furbish et al., Proc. Natl. Acad. Sci. USA74, 3560-3563 (1977); Dale and Beutler, Ibid. 73, 4672-4674 (1976)].Glucocerebrosidase activity was measured by adding enzyme (5-50 μl) to asonicated suspension of buffer (50 μl of 50 mM sodium citrate/sodiumphosphate buffer, pH 5.0) containing glucosyl ceramide (1 mM), Triton®X-100 non-ionic surfactant (0.25% v/v) and sodium taurodeoxycholate(0.6% v/v) that had been previously dried under nitrogen fromchloroform:methanol (2:1 v/v) solutions. After incubation at 37° C. for15-60 min., the reaction was stopped by the addition of 500 μl ofchloroform:methanol and the phases separated by centrifugation. Theupper phase was washed twice with Folch theoretical lower phase [Folchet al., J. Biol. Chem. 226, 497-509 (1957)] desalted using AG50-X12ion-exchange resin and dried under vacuum. The reaction products wereseparated by high performance anion exchange chromatography (DionexBioLC System) and detected by pulsed amperometry. The amount ofenzyme-released glucose was calculated from peak areas by applyingexperimentally determined response factors for glucose relative to anincluded reference monosaccharide [Butters et al, Biochem. J. 279,189-195 (1991)].

UDP-glucose:N-acylsphingosine glucosyltransferase (EC 2.4.1.80) activitywas measured in rat brain homogenates and mouse macrophage tissuecultured cell (WEHI-3B) homogenates using a method adapted as followsfrom published conventional procedures [Vunnam and Radin, Chem. & Phys.of Lipids 26, 265-278 (1980); Shukla and Radin, Arch Biochem. Biophys.283, 372-378 (1990)]: Dioleoylphosphatidylcholine and cerebrosideliposomes containing 200 nmol ceramides Type IV (Sigma) were added to areaction mixture (100 μl) composed of 40 mM2-[N-morpholino]ethanesulfonic acid (MES) buffer, pH 6.5, 5 mM MnCl₂,2.5 mM MgCl₂, 1 mM NADH and 8 μM UDP-[¹⁴ C]-glucose (318 mCi/mmol,Amersham International, Amersham, U.K.). After incubation at 37° C. for1-2 hr. the reaction was stopped by the addition of EDTA (25 mM) and KCl(50 mM). Radiolabelled glycolipids were extracted with 500 μl ofchloroform:methanol (2:1 v/v) for 10 min. and the phases separated. Thelower phase was washed twice with Folch theoretical upper phase andportions taken for scintillation counting. When imino sugars were testedfor inhibitory activity, these were added at appropriate concentrationsto homogenates and preincubated for 10 min. before sonication withceramide containing liposomes. Control reactions were performed withliposomes containing no ceramide to measure the activity of transfer toendogenous acceptors.

Glycolipid Analysis:

HL-60 cells were cultured by conventional procedure as previouslydescribed by Platt et al., Eur. J. Biochem. 208, 187-193 (1992). HL-60cells at 5×10⁴ cells/ml were cultured in the presence or absence ofimino sugars for 24 hr. For labelling, the two dimensional thin layerchromatography (2D-TLC) conventional method of Butters and Hughes wasfollowed [In Vitro 17, 831-838 (1981)]. Briefly, [¹⁴ C]-palmitic acid(56.8 mCi/mmol, ICN/Flow) was added as a sonicated preparation in foetalcalf serum (FCS, Techgen, London, U.K., 0.5 μCi/ml) and the cellscultured for a further 3 days maintaining the imino sugars in themedium. The cells were harvested, washed three times with phosphatebuffered saline (PBS), extracted in 1 ml chloroform:methanol (2:1 v/v)and separated by 1 dimensional TLC, loading equal counts (1D-TLC,chloroform:methanol:water (65:25:4)). For two dimensional separationsthe one dimensional separation was performed as described above, theplate dried overnight under vacuum and separated in the second dimensionusing a solvent of tetrahydrofuran: dimethoxymethane:methanol:water(10:6:4:1), Plates were air dried and exposed to Hyperfilm-MP highperformance autoradiography film (Amersham).

Cell Culture and Metabolic Labelling:

The culture of CHO cells expressing soluble recombinant gp120 (from Dr.P. Stevens, MRC AIDS Directed Programme Reagent Project) and theradiolabelling of these cells was carried out by conventional procedureas described by Karlsson et al., J. Biol. Chem. 268, 570-576 (1993).Briefly, CHO cells were harvested mechanically, washed three times withphosphate buffered saline, 0.1M pH 7.2 (PBS) and resuspended inmethionine- and cysteine-free RPMI-1640 medium (ICN-Flow Laboratories,High Wycombe, Bucks, U.K.) supplemented with 1% dialysed FCS. Cells (10⁷/ml) were preincubated in the presence or absence of NB-DNJ or NB-DGJfor 1 hr prior to the addition of 100 μCi/ml Tran³⁵ S-label (ICN-Flow)for 4 hr. The supernatants were collected and concentrated tenfold usinga 30 kDa cut-off membrane (Amicon, Danvers, Mass., U.S.A. ).

Immunoprecipitation:

Immunoprecipitations were performed by conventional procedure asdescribed by Karlsson supra. Supernatants were incubated with the mAbABT 1001 monoclonal antibody (American Biotechnologies Inc., Cambridge,Mass., U.S.A.) at 0.5 μg/100 μl of supernatant for 30 min. at roomtemperature followed by sheep anti-mouse IgGl-coated magnetic beads(Dynal Ltd., Wirral, Merseyside, U.K., 1.2×10⁷ beads per sample) for 1hr. at 4° C. The beads were washed three times with 2% Triton® X-100 inPBS and three times with PBS. Gp120 was eluted in 100 μl reducingSDS-PAGE sample buffer with heating (95° C., 5 min.). Each sample wasdivided into two equal aliquots and 25 μl of dH₂ O added to give a finalvolume of 50 μl. To one half of each sample 2 μl of endoglycosidase H(endo H, 1 unit/ml, Boehringer Mannheim Ltd., Lewes, Sussex, U.K.) wasadded and the other half left untreated. Digestion was performed at 37°C. for 18 hr. and terminated by the addition of 50 μl of SDS-PAGEreducing sample buffer (95° C., 5 min.).

Glycopeptide Analysis:

HL-60 and BW5147 cells were cultured in RPMI-1640 and 10% FCS. The cellswere incubated for 30 min. in the presence or absence of 2 mM NB-DNJ orNB-DGJ in reduced glucose RPMI-1640 medium (Flow), supplemented with 1%dialysed FCS. [³ H]-mannose (16.5 Ci/mmol, Amersham) was added at 200μCi/ml and the cells cultured for a further 3 hr. Washed cell pelletswere resuspended in 50 mM TrisHC1 buffer, pH 7.5, containing 10 mM CaCl₂and 0.02% sodium azide and heated at 100° C. for 5 min. After cooling,Pronase® enzyme was added to 0.04% (w/v final concentration) andincubated for 96 hr. at 37° C. under toluene with aliquots of Pronase®added at each 24 hr. period. The digestion was stopped by boiling for 5min., and glycopeptides recovered by centrifugation at 13000 g for 10min. Samples were fractionated by Con A-Sepharose® chromatographyaccording to conventional procedure of Foddy et al., Biochem. J. 233.,697-706 (1986).

In Vitro Gaucher's Disease Model:

The in vitro Gaucher's disease model was prepared as follows: WEHI-3Bcells (American Type Culture Collection, Rockville, Md., U.S.A.) weremaintained in logarithmic phase growth for 14 days in RPMI-1640 medium,10% FCS, in the presence or absence of 50 μM conduritol β epoxide (CBE,Toronto Research Chemicals, Downsview, Canada) with or without NB-DNJ orNB-DGJ. Cells were passaged every 3 days and compound concentrationsmaintained throughout. Equal cell numbers (5×10⁶) were harvested,extracted in 1 ml chloroform: methanol (2:1 v/v) overnight at 4° C., theextracts centrifuged, the chloroform:methanol extract retained and thepellet re-extracted as above for 2 hr. at room temperature. Pooledextracts were dried under nitrogen, re-dissolved in 10 μlchloroform:methanol (2:1 v/v) and separated by 1D-TLC inchloroform:methanol:water (10:6:4:1). Plates were air dried andvisualized using α-naphthol (1% w/v in methanol) followed by 50% (v/v)sulphuric acid.

Transmission Electron Microscopy:

Cells for electron microscopy were harvested (1×10⁷ cells pertreatment), washed three times in serum free RPMI-1640 medium and fixedin medium containing 2% glutaraldehyde (v/v) and 20 mM Hepes (v/v) onice for 2 hr. Cells were washed in 0.1 M cacodylate buffer containing 20mM calcium chloride (w/v). Cells were post-fixed with 1% osmiumtetroxide in 25 mM cacodylate buffer (w/v) containing 1.5% potassiumferrocyanide (w/v) for 2 hr. on ice. Samples were dehydrated through anethanol series, transferred to propylene oxide and embedded in Embed 800(Electron Microscopy Sciences, Pa., U.S.A.). The sections were stainedwith uranyl acetate/lead citrate and observed with a Hitachi 600microscope at 75 kv.

Analysis of cholera toxin binding to the H9 human lymphoid cell linefollowing three days treatment with NB-DNJ or NB-DGJ:

Methods: Cells were maintained in logarithmic phase growth in RPMI-1640medium. Cholera toxin B chain (Sigma) was conjugated to fluoresceinisothiocyanate (Sigma) and flow cytometric analysis was carried out byconventional procedure as described by Platt et al., Eur. J. Biochem.208, 187-193 (1992). Analysis was performed on a FACScan Cytometer(Becton Dickinson, Sunnyvale, Calif., USA). Data on viable cells werecollected on a four decade logio scale of increasing fluorescenceintensity. The data are presented as percent reduction in cholera toxinbindings sites per cell on the y-axis against compound concentration onthe x-axis. The specificity of cholera toxin: cell surface binding wasestablished by inhibiting this interaction with a one hundred fold molarexcess of GMI derived oligosaccharide,GalβGalNAcβ4(NeuAcα3)Galβ4Glcβ3Cer.

Results

Comparison of N-alkylated imino sugars as inhibitors of glycolipidbiosynthesis.:

The glucose analogue, NB-DNJ and four pyranose analoguss (NB-DMJ,mannose analogue; NB-DFJ, fucose analogue; NB-DGJ, galactose analogue;and NB-NAG, N-acetylglucosamine analogue) were assessed by the abovemethods for their capacities to inhibit the metabolic incorporation ofradiolabelled palmitate into glycolipids in HL-60 cells at a 500 μMcompound concentration using 1D-TLC analysis (FIG. 1). In addition toNB-DNJ, the only analogue which specifically inhibited glycolipidbiosynthesis was NB-DGJ. All other analogdes were without effect. BothNB-DNJ and NB-DGJ inhibited the biosynthesis of Glc-Cer, gangliosidesand an unknown lipid species in agreement with the previous observationswith NB-DNJ described in copending application Ser. No. 08/061,645. Toconfirm that NB-DNJ and NB-DGJ had comparable effects on the completespectrum of glycolipids in this cell line, 2D-TLC was performed toresolve further the individual glycolipid species (FIG. 2). A totaldepletion of glycolipid species was achieved with both 500 μM NB-DNJ andNB-DGJ. Specifically, gangliosides, the unknown lipid (N*) and both themono and dihexaside species were absent following treatment with eithercompound. Phospholipid composition and relative abundance werecomparable, irrespective of treatment, consistent with the previousobservations in copending application Ser. No. 08/061,645 thatN-alkylated imino sugars have no effect on sphingomyelin or phospholipidbiosynthesis. When the two analoguss were compared at a range ofconcentrations by 1D-TLC (FIG. 3) both analogdes exhibited completeglycolipid inhibition between 50 μM and 500 μM concentrations, althoughpartial inhibition occurred with both compounds at concentrations as lowas 0.5-5 μM. Both analoguss were non-cytotoxic in the dose range tested.

Effects of increasing DNJ and DGJ N-alkyl chain length on inhibition ofglycolipid biosynthesis:

A series of N-alkylated DNJ and DGJ derivatives were compared for theirabilities to inhibit glycolipid biosynthesis (FIG. 4A and 4B,respectively) by 1D-TLC. The non-alkylated imino sugars and the N-methylDNJ, N-methyl DGJ and N-ethyl DGJ had no effect on glycolipidbiosynthesis. The N-propyl analogues of both parent compounds showedpartial inhibitory activity, whereas the N-butyl and N-hexyl derivativesof DNJ and DGJ completely inhibited glycolipid biosynthesis, asdetermined by the loss of detectable Glc-Cer. These data were thereforein agreement with the data from the previous application Ser. No.08/061,645 (where the N-methyl derivative was compared with N-butyl andN-hexyl DNJ). There is a minimal N-alkyl chain length requirement toachieve full inhibition of glycolipid biosynthesis, with butyl and hexylbeing optimal.

Analysis of NB-DGJ in an in vitro Gaucher's disease model:

The WEHI-3B murine macrophage cell line can be induced to resembleGaucher's cells by treatment with the irreversible glucocerebrosidaseinhibitor CBE. NB-DNJ and NB-DGJ were compared in their ability toprevent the accumulation of Glc-Cer in this system (FIG. 5). Bothanalogues prevented CBE induced glycolipid storage in the 5-50 μM doserange. These data therefore demonstrate that NB-DGJ is as effective asNB-DNJ in preventing glycolipid storage in this in vitro Gaucher'sdisease model. The status of the lysosomes from cells treated witheither NB-DNJ or NB-DGJ was assessed by transmission electron microscopy(FIG. 6). It was found that both analogues prevented the glycolipidaccumulation observed in the lysosomes of cells treated with CBE.

Specificity of NB-DGJ for the glycolipid biosynthetic pathway:

The CHO cell line is unique in that it lacks significant levels of theGolgi endomannosidase which acts to circumvent α-glucosidase I and IIinhibition [Karlsson et al., J. Biol. Chem. 268, 570-576 (1993);Hiraizumi et al., J. Biol. Chem. 268, 9927-9935 (1993)]. As aconsequence, it offers an unambiguous cellular system in which to testα-glucosidase inhibition. NB-DNJ was previously tested in this cell lineexpressing recombinant gp120 and it was found that it results in themaintenance of glucosylated high mannose oligosaccharides on gp120 whichare fully sensitive to endo H [Karlsson et al., supra].

Analysis of the N-linked oligosaccharides of gp120 expressed in CHOcells was performed in the presence or absence of NB-DNJ or NB-DGJ (FIG.7). Treatment of CHO cells with 0.5 mM or 5 mM NB-DNJ resulted in fullyendo H sensitive gp120 N-linked glycans in contrast to the untreatedgp120 which was partially sensitive to endo H. This partial sensitivityof untreated gp120 to endo H is because gp120 carries approximatelyfifty percent high mannose N-linked oligosaccharides per molecule[Mizuochi et al., Biochem. J. 254, 599-603 (1988); Mizuochi et al.,Biomed. Chrom. 2, 260-270 (1988)]. However, when the galactose analogue,NB-DGJ, was tested in this system, at 0.5 mM and 5 mM concentrations,gp120 remained partially sensitive to endo H and was indistinguishablefrom the untreated gp120 molecules. This suggested that the galactoseanalogue was not acting as an inhibitor of α-glucosidases I and II.

To examine the effect on endogenous glycoprotein synthesis,radiolabelled glycopeptides were isolated from treated HL-60 and murineBW5147 cells and analysed for their affinity for Con A-Sepharose®. Thisprocedure efficiently resolves tetra- and tri-antennary complexN-glycans from bi-antennary and high mannose/hybrid N-glycans [Foddy etal., Biochem J. 233, 697-706 (1986)]. Addition of NB-DNJ changes theaffinity of glycopeptides eluting from the Con A-Sepharose® column(Table 4) as a result of processing glucosidase inhibition. Thus theproportion of unbound glycans (tetra- and tri-antennary species)decreases, and a corresponding increase is found in the proportion ofhigh mannose/hybrid glycans that are tightly bound to Con A-Sepharose®and eluted with 500 mM methylmannoside. Similar gross changes inglycopeptide composition following treatment with α-glucosidaseinhibitors are well established [Moore and Spiro, J. Biol. Chem. 265,13104-13112 (1990)]. The galactose analogue, NB-DGJ, showed an unchangedglycopeptide profile by Con A-Sepharose® chromatography (Table 4). Toconfirm these data, glucosidase inhibition was measured directly invitro using a mixture of purified α-glucosidases I and II (FIG. 8).Whereas NB-DNJ inhibited glucosidase I and II with an IC₅₀ of 0.36 μM,NB-DGJ was only weakly inhibitory (IC₅₀ of 2.13 mM, Table 5). These dataprovide substantial evidence that in both in vitro α-glucosidase assaysand in intact cellular system assays NB-DGJ does not inhibit N-linkedoligosaccharide processing.

DNJ and its N-alkylated derivatives are inhibitors of the purifiedlysosomal glucocerebrosidase enzyme required for the cleavage of Glc-Certo glucose and ceramide [Osiecki-Newman et al., Biochim. Biophys. Acta.915, 87-100 (1987)]. In recent tests with the in vitro Gaucher's diseasemodel in co-pending application Ser. No. 08/061,645, it was observedthat WEHI-3B cells incubated in the absence of CBE but in the presenceof NB-DNJ accumulated Glc-Cer. It was therefore apparent that theN-butyl derivative of DNJ was also acting as an inhibitor ofglucocerebrosidase in a cellular environment. The inhibitory activity ofNB-DNJ and NB-DGJ was therefore directly measured to investigatequantitatively their capacities to inhibit human placentalglucocerebrosidase (Table 5). NB-DNJ provided moderate inhibition ofcatalysis with an IC₅₀ of 0.52 mM while NB-DGJ did not inhibit enzymeactivity even at the highest concentration tested (1 mM). In terms ofpercent enzyme inhibition achieved with the two analogues, 1 mM NB-DNJresulted in 90% inhibition while 1 mM NB-DGJ was non-inhibitory (FIG.8), thereby further confirming the advantageous and unexpected selectiveinhibitory activity of NB-DGJ compared to that of NB-DNJ.

Inhibition of UDP-glucose: N-acylsphingosine glucosyltransferase:

The determination of transferase activity using rat brain or mousemacrophage tissue cultured cells followed saturation kinetics for bothexogenously added ceramide acceptor and UDP-glucose donor. Under theseconditions both N-butylated DNJ and DGJ were moderate inhibitors ofglucose transfer, (IC₅₀ 2.95 mM and 60.88 respectively, Table 5) whereastheir unmodified parent homologues were not inhibitory at the highestconcentration tested 6.1 and 5.0 mM, respectively, FIG. 8).

Analysis of cholera toxin binding to the H9 human lymphoid cell linetreated with NB-DGJ:

The activity of the representative N-butyl deoxygalactonojirimycin(NB-DGJ) for inhibiting the surface expression of glycolipid receptorsfor bacteria and bacterial toxins was illustrated by subjecting H9 cellsto cholera toxin binding sites in the presence of varying concentrationsof the NB-DGJ. As a specific probe, advantage was taken of the GM1binding specificity of the cholera toxin B chain [van Heyningen, Nature249, 415-417 (1974); Karlsson, Ann. Rev. Biochem. 58, 309-350 (1989)].The binding of cholera toxin to H9 cells cultured in the presence ofNB-DGJ was reduced by approximately 70% (FIG. 11). This was consistentwith the loss of GM1 from the cell surface and provided further evidencefor the inhibition of glycolipid biosynthesis by NB-DGJ, even though bycomparison it was less than the approximately 90% reduction (FIG. 11)obtained with the N-butyl deoxynojirimycin (NB-DNJ). These resultsdemonstrate that the imino sugar derivatives have use as anti-microbialagents by inhibiting the surface expression of glycolipid receptors forbacteria and bacterial toxins as shown in Tables 1 and 2, respectively.

                  TABLE 1                                                         ______________________________________                                        GLYCOSPHINGOLIPID RECEPTORS FOR BACTERIAL                                     CELLS                                                                         Microorganism                                                                              Target Issue                                                                             Presumed Specificity                                  ______________________________________                                        E. coli      Urinary    Galα4Galβ                                  E. coli      Urinary    GlcNAcβ                                          Propionibacterium                                                                          Skin/Intestine                                                                           Galβ4Glcβ                                   Several bacteria                                                                           Diverse    Galβ4Glcβ                                   Streptococcus                                                                              Respiratory                                                                              GlcNAcβ3Gal                                      pneumoniae                                                                    E. coll CFA/I                                                                              Intestine  NeuAcα8                                         E. coli      Urinary    NeuAcα3Gal                                      E. coli      Intestine  NeuGcα3Galβ4GlcβCer                                           GalNAcβ4 (NeuAcα3)-                                                Galβ4GlcβCer                                Staphylococcus                                                                             Urinary    Galβ4GlcNAc                                      saprophyticus                                                                 Actinomyces naeslundi                                                                      Mouth      Galβ, GalNAcβ,                                                      Galβ3GalNAcβ,                                                       GalNacβ3Galβ                                Pseudomonas  Respiratory                                                                              GalNAcβ4Gal                                      Neisseria gonorrhoeae                                                                      Genital    Galβ4Glcβ                                                           NeuAcα3Galβ4GlcNAc                         ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        GLYCOSPHINGOLIPID RECEPTORS FOR BACTERIAL                                     TOXINS                                                                                                       Presumed                                                             Target   Receptor                                       Microorganism                                                                           Toxin       Tissue   Sequence                                       ______________________________________                                        Vibrio cholerae                                                                         Cholera toxin                                                                             Small    Galβ3GalNAcβ4-                                             Intestine                                                                              (NeuAcα3)Gal-                                                           β4GlcβCer                            E. coli   Heat-labile Intestine                                                                              Galβ3GalNAcβ4-                                 toxin                (NeuAcα3)Gal-                                                           β4GlcβCer                            Clostridium                                                                             Tetanus toxin                                                                             Nerve    Galβ3GalNAcβ4-                       tetani                         (NeuAcα8Neu-                                                            Acα3)Galβ4Glc-                                                     βCer                                      Clostridium                                                                             Botulinum   Nerve    NeuAcα8NeuAcα-                     botulinum toxin A and E                                                                             Mem-     3Galβ3GalNAcβ-                                             brane    4(NeuAcα8Neu-                                                           Acα3)Galβ4Glc-                                                     βCer                                      Clostridium                                                                             Botulinum   Nerve    GalNAβ4(Neu-                              botulinum toxin B, C  Mem-     GalNAβ4(Neu-                                        and F       brane    Acα8NeuAcα3)-                                                     Galβ4GlcβCer                         Clostridium                                                                             Botulinum   Nerve    GalβCer                                   botulinum toxin B     Mem-                                                                          brane                                                   Clostridium                                                                             Delta toxin Cell lytic                                                                             GalNAcβ4-                                 perfringens                    (NeuAcα3)Galβ-                                                     4GlcβCer                                  Clostridium                                                                             Toxin A     Large    Galα3GalβGlc-                       difficile             Intestine                                                                              NAcβ3Galβ4-                                                         GlcβCer                                   Shigella  Shiga toxin Large    Galα4GalβCer                        dysenteriae           Intestine                                                                              Galα4Galβ4Glc-                                                     βCer                                                                     GlcNAcβ4Glc-                                                             NAc                                            E. coli   Vero toxin or                                                                             Intestine                                                                              Galα4Galβ4-                                   Shiga-like           GlcβCer                                             toxin                                                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        HERIDITARY GLYCOLIPID STORAGE DISORDERS                                       Disease   Lipid Accumulation                                                                           Enzyme Defect                                        ______________________________________                                        Gaucher's Glucocerebroside                                                                             Glucocerebroside-β-                                                      glucosidase                                          Ceramide  Ceramide Lactoside                                                                           Ceramidelactoside-β-                            Lactoside                galactosidase                                        Lipidosis                                                                     Fabry's   Ceramide Trihexoside                                                                         Ceramidetrihexoside-α-                                                  galactosidase                                        Tay-Sach's                                                                              Ganglioside GM2                                                                              Hexosaminidase A                                     Sandhoff's                                                                              Globoside and GM2                                                                            Hexosaminidase A and B                               General   Ganglioside GM1                                                                              β-Galactosidase                                 Gangliosidosis                                                                Fucosidosis                                                                             H-isoantigen   α-Fucosidase                                   Krabbe's  Galactocerebroside                                                                           Galactocerebroside-β-                                                    galactosidase                                        Metrachromatic                                                                          Sulfatide      Sulfatidase                                          Leukodystrophy                                                                ______________________________________                                    

                                      TABLE 4                                     __________________________________________________________________________    EFFECT OF IMINO SUGAR ANALOGUES ON OLIGO-                                     SACCHARIDE BIOSYNTHESIS                                                                                     Total .sup.3 H-                                             Tetra-&      Oligo-                                                                             mannose                                                     Tri-  Bi-    mannose                                                                            recovered                                       Cell line                                                                          Treatment                                                                            antennary                                                                           antennary                                                                            & hybrid                                                                           (cpm)                                           __________________________________________________________________________    HL-60                                                                              untreated                                                                            28.3  19.7   53.0 666918                                               NB-DNJ 19.5  20.0   60.5 913095                                               NB-DGJ 29.1  17.7   54.2 844322                                          BW5147                                                                             untreated                                                                            46.1  5.6    48.3 476527                                               NB-DNJ 26.8  4.9    69.3 686026                                               NB-DGJ 40.4  7.2    52.4 706873                                          __________________________________________________________________________

Cells were radiolabelled for 4 hr. with [³ H]-mannose in the presence orabsence of compounds as shown above. Washed cells were exhaustivelydigested with Pronase® enzyme and resultant glycopeptides fractionatedby Con A-Sepharose® chromatography as described hereinbefore. Thepercentage of radiolabelled glycopeptides that were non-bound (complextetra- and tri-antennary N-glycans), eluted with 10 mM methylglucoside(complex bi-antennary N-glycans), or further eluted with 500 mMmethylmannoside (oligomannose and hybrid N-glycans) were calculated fromestimations of radioactivity recovered from pooled eluates.

                  TABLE 5                                                         ______________________________________                                        CONCENTRATIONS OF IMINO SUGAR ANALOGUES                                       REQUIRED FOR THE INHIBITION OF GLYCOLIPID                                     AND GLYCOPROTEIN METABOLISING ENZYMES                                                     Compound IC.sub.50 values                                         Enzyme        DNJ      NB-DNJ   DGJ  NB-DGJ                                   ______________________________________                                        UDP-glucose:N-                                                                              --†                                                                             2.95 mM  --†                                                                         60.88 mM                                 acylsphingosine                                                               glucosyltransferase                                                           β-glucocerebrosidase                                                                   2.43 mM  0.52 mM  --*  --*                                      α-glucosidase I and II                                                                nd       0.36 μM                                                                             nd    2.13 mM                                 ______________________________________                                         *not inhibitory at 1 mM concentrations of compound.                           † not inhibitory at the highest concentration tested (see FIG. 8)      nd not determined                                                        

Enzymes were assayed according to procedure described hereinbefore usingconcentrations of analogues shown in FIG. 8. The data from FIG. 8 wereplotted on a logarithmic scale for accurate estimations of IC₅₀ values,shown above.

In addition to their use as inhibitors of glycolipid biosynthesis incells, the inhibitory agents described herein also can be used foradministration to patients afflicted with glycolipid storage defects byconventional means, preferably in formulations with pharmaceuticallyacceptable diluents and carriers. These agents can be used in the freeamine form or in their salt form. Pharmaceutically acceptable saltderivatives are illustrated, for example, by the HCl salt. The amount ofthe active agent to be administered must be an effective amount, thatis, an amount which is medically beneficial but does not present toxiceffects which overweigh the advantages which accompany its use. It wouldbe expected that the adult human daily dosage would normally range fromabout one to about 100 milligrams of the active compound. The preferableroute of administration is orally in the form of capsules, tablets,syrups, elixirs and the like, although parenteral administration alsocan be used. Suitable formulations of the active compound inpharmaceutically acceptable diluents and carriers in therapeutic dosageform can be prepared by reference to general texts in the field such as,for example, Remington's Pharmaceutical Sciences, Ed. Arthur Osol, 16thed., 1980, Mack Publishing Co., Easton, Pa., U.S.A.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed:
 1. The method of inhibiting the surface expression ofglycolipid receptors for bacteria and bacterial toxins comprisingsubjecting cells susceptible to said expression to an effective amountfor inhibiting said expression of an N-alkyl derivative ofdeoxygalactonojirimycin in which said alkyl contains from 3-6 carbonatoms.
 2. The method of claim 1 in which the alkyl group contains from4-6 carbon atoms.
 3. The method of claim 2 in which the alkyl group isbutyl.
 4. The method of claim 2 in which the alkyl group is hexyl. 5.The method of claim 1 in which the inhibitory effective amount is fromabout 50 μM to about 500 μM.
 6. The method of claim 1 in which thebacterial toxin is cholera toxin.